Background: During calcific aortic valve disease (CAVD) progression, mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, which further disrupts cellular-driven pathophysiology and valve biomechanics. Currently, no pharmacotherapeutics are available for CAVD, due in part to the lack of 1) appropriate experimental models that recapitulate this complex biomechanical environment; and 2) studies that adequately assess the complexity of novel engineered valvular model systems.

Methods: Three models of CAVD were used in this study: 1) traditional 2D- valve interstitial cell (VIC) monoculture; 2) VICs encapsulated in 3D-bioprinted models; and 3) human CAVD valve leaflets. Multi-omics LC-MS/MS analysis were performed to probe the cellular proteome, extracellular matrisome, vesiculome, and n-glycome.

Results: Cellular proteomics identified over 2500 proteins. The 2D-cellular proteome was enriched in biological processes of actin organization and platelet aggregation, while the 3D-bioprinted cellular proteome was enriched in glycosaminoglycan biosynthesis, and extracellular-matrix organization. 3D-printed cellular proteomics also identified enzymatic regulators of glycosylation, informing downstream N-glycome composition. Extracellular vesiculomics identified over 200 proteins, including 14 vesicle markers. Additionally, extracellular-matrisomics probed site-specificity and abundance of several post-translational modfications of collagen subtypes.

Conclusion: This study positions multi-omics as a novel technique for the design and assessment of bioengineered model systems.